Nucleic acids such as DNA and RNA are important biomolecules involved in genetic information storage and transmission from one generation to the next and in the routine functioning of all living organisms. As a result, nucleic acid detection is frequently conducted in life science research labs and has many practical applications, ranging from species identification to genetic disease screening, to pathogen detection and to forensic science, merely by way of example.
Among the different methods of nucleic acid detection, nucleic acid binding dyes are widely used because of the simplicity and high sensitivity offered (Deligeorgiev, et al. Recent Patents on Materials Science 2, 1-26(2009)). Nucleic acid binding dyes can be intrinsically nonfluorescent or only weakly fluorescent by themselves but may become highly fluorescent upon binding to nucleic acids, and thus may provide a high signal-to-noise ratio during detection. Binding of the dyes may take the mode of intercalation, where the dye may insert itself in between two adjacent nucleic acid base pair, or it may take the mode of minor groove binding, where the dye may reside in the minor groove of a double-stranded nucleic acid. Accordingly, the dyes may be termed as intercalators or minor groove binders, depending on the binding mode. Nucleic acid dyes may be extensively used in various practical applications. Some non-limiting examples may include nucleic acid detections, such as real-time qPCR for gene detection, visualization of cell nuclei, quantification of total nucleic acids in solutions, and staining of nucleic acids in gel matrix.
While nucleic acid dyes are highly useful, they may also pose serious safety issues for people who handle and dispose of them. Such issues may arise from the very DNA-binding nature of the dyes because the dyes' interaction with nucleic acids can interfere with the replication, transcription and translation of nucleic acids in cells (MacCnan, et al. Proc Natl Acad Sci USA 72(12), 5135(1975)). Moreover, nucleic acid dyes may greatly potentiate the DNA mutation caused by UV light or other known mutagens (Ohta et al. Mutation Research 492(1-2), 91(2001)). For these reasons, nucleic acid dyes can be potential mutagens and/or carcinogens. The safety issue is of special concern to nucleic acid gel staining because the amount of the dye solution used in the experiments may be large, for example, as much as 50 mL or higher, and because the experiments are routinely performed in many labs. One widely used gel stain is ethidium bromide, which is a known mutagen and highly suspected carcinogen (National Toxicology Program (Aug. 15, 2005). “Executive Summary Ethidium Bromide: Evidence for Possible Carcinogenic Activity”). Other dyes such as SYBR Safe, which have been alleged to be less mutagenic than ethidium bromide in Ames tests, may not represent an adequate solution because the increased cytotoxicity of SYBR Safe relative to ethidium bromide may make an assessment of SYBR Safe's mutagenicity at higher dye concentration difficult (SYBR Safe white paper, Life Technologies, 2003). Other approaches to improve safety have relied on the large physical size of the dyes without compromising the dyes' performance as gel stains. It is believed that the large molecular size may limit the cell membrane permeability of the dyes, thus reducing the chance for the dyes to interact with and hence cause damage to genomic DNA. Although such dyes may work very well in agarose-based nucleic acid gel staining, their large size may impede the dyes' diffusion into the more densely cross-linked polyacrylamide gels (PAGE gels) used for analyzing small nucleic acid fragments, and can result in relatively weak staining. Thus, there remains a need for safe and sensitive nucleic acid gel stains that overcome the drawbacks of the existing dyes.